Cyclodextrins are cyclic molecules consisting of 1-4 linked alpha-D-glucopyranose monomeric units. The cyclodextrins containing 6-, 7-, and 8-glucose units joined to form a ring, commonly known as alpha-, beta-, and gamma-cyclodextrin, respectively, are the most important cyclodextrins to date, possibly because of their availability relative to cyclodextrins of different ring size. The usefulness of these cyclodextrins arises from their ability to reversibly form inclusion complexes, or clathrates, with many types of compounds. Inclusion complexes arise when a host molecule, such as a cyclodextrin, has a structure containing an interior cavity into which guest molecules can bind by weak interactions such as van der Waal's forces. The latter are short range forces which are sufficiently strong to allow the formation of definite, generally solid complexes, but are sufficiently weak to permit ready dissociation of the complex to a host and guest molecule.
The cyclodextrins are doughnut-shaped molecules with an interior cavity whose size and shape is determined by the number of glucose units that make up the ring. In alpha-cyclodextrin the almost cylindrical cavity is approximately 7 angstroms deep and 5 angstroms in diameter. In beta-cyclodextrin the depth is the same but the diameter is 7 angstroms, and in gamma-cyclodextrin cavity is again 7 angstroms deep but is 9 angstroms in diameter. Cyclodextrins are soluble in water because of the many hydroxyl groups of the glucose subunits that surround the rim of the cavity. However, the interior of the cavities themselves is hydrophobic, and these hydrophobic cavities extract organic molecules from aqueous solution if the organic materials have the correct shape and hydrophobic character.
The complexing ability of cyclodextrins lends itself to various uses. For example, the cyclodextrins are used in encapsulating desirable flavors and fragrances which can then be stored for reasonably long periods of time and added to foods at their preparation. Reciprocally, cyclodextrins may be used in removing undesirable flavors and fragrances from food by complexing with them. Cyclodextrins also are used in the protection of foods against oxidation, photochemical degradation, and thermal decomposition. These and other uses have been summarized by J. Szejtli, Starch, 34, 379-385 (1982)
Commercial utilization of cyclodextrins has been impeded by their relatively high cost resulting from current process limitations. Cyclodextrins generally are formed in relatively poor yield from starch. The necessity of using feedstocks with a relatively low solids content further limits productivity and affords a dilute solution of cyclodextrins which additionally complicates their isolation. Although a method of enhancing cyclodextrin production as recently described in Ser. No. 36,725 ameliorates the cost somewhat, there remain limitations associated with the enzyme itself.
In particular, although cyclodextrin glycosyltransferases are available from several sources, apparently that from Bacillus circulans finds greatest commercial use. However, this particular source of the enzyme is unsatisfactory in many regards. Perhaps its greatest disadvantage is that a complex, expensive medium is needed for its adequate growth and enzyme production, although this is not its sole disadvantage.
The inadequacies of known microorganisms as cyclodextrin glycosyltransferase producers was the incentive for our seeking a microorganism which would prove superior. The criteria which needed to be satisfied by a microorganism were decided upon by pragmatic considerations relating to the cost and ease of enzyme production. Among the characteristics sought in the microorganism is rapid growth in simple media. The microorganism also was required to produce high levels of enzyme extracellularly over a broad pH range, which translates to relative insensitivity of enzyme production to changing culture conditions. It also was necessary that the enzyme be produced under conditions where it could be easily separated and purified.
In this application are described a trio of cyclodextrin glycosyltransferase producers isolated from different soil samples. Two are bacilli each elaborating an enzyme producing largely .beta.-cyclodextrin. The remaining microorganism is gram negative and elaborates an enzyme producing comparable amounts of both .alpha.- and .beta.-cyclodextrin. Differences in the phenomenological characteristics of cyclodextrin glycosyltransferases are well known, with enzymes appearing to fall in one of the foregoing two classes. The microorganisms described herein grow quite well on very simple, inexpensive media, are relatively insensitive to pH of the medium, and show good enzyme production at an early growth stage even in simple media. Since enzyme production is recognized as being responsive to changes in the growth medium, it will be appreciated that the enzyme production levels referred to herein are not optimized and may represent only a fraction of that which the microorganism is capable of producing under optimum conditions.
The sensitivity of some of the prior art cyclodextrin glycosyltransferase producers to growth medium pH is well documented. Among the outstanding successes of our invention is achievement of our goal to find such producers having the ability to grow well over a relatively broad range of pH and to grow rapidly even in simple media.